Transcript analysis of heat shock protein 72 in vitrified 2-cell mouse embryos and subsequent in vitro development.

OBJECTIVE
The aim of the study was to compare the effects of two different concentrations of cryoprotectants by cryotopvitrification on survival, developmental capacity and Heat shock protein 72 (Hsp72) expression of two-cell mouse embryos.


MATERIALS AND METHODS
In this experimental study, transcript analysis of Hsp72 gene was performed on non-vitrified and vitrified 2-cell mouse embryos via a nested quantitative polymerase chain reaction (nqPCR) subsequent to normalization with Hprt1 as the reference gene. The different cryoprotectant combinations were 15% (vit1:7.5% of each ethylene glycol (EG) and dimethyl sulfoxide (DMSO), 30% (vit2:15% EG + 15% DMSO) and control group with no cryoprotectants. Vitrified and fresh 2-cell embryos were cultured to obtain cleavage and blastocyst formation rates. The results were analyzed via one-way analysis of variance and the mean values were compared with least significant difference (LSD) (p< 0.05).


RESULTS
The relative expression of Hsp72 in vit2 (30% v/v) was significantly higher than vit1 (15% v/v). Survival rates were the same for both vitrification treatments and significantly lower than the control group. Cleavage and blastocyst rates in vit1 were significantly higher than vit2 while those in two vitrified groups were significantly lower than the control group.


CONCLUSION
Our developmental data demonstrated that vit1 treatment (7.5% EG and 7.5% DMSO) was more efficient than vit2 (15% EG and 15% DMSO) in mouse embryos. The cryotopvitrification with two concentrations of cryoprotectants caused the relative changes of Hsp72 transcript level, but the stability of the gene in vit1 was significantly higher than vit2 and closer to the fresh 2-cell embryos.


Introduction
In assisted reproduction, embryo cryopreservation has proven to be a powerful tool with applications in bioscience, agriculture and medicine (1). It has been demonstrated that ovarian hyperstimulation syndrome can be decreased via embryo cryopreservation.
Additionally, it has been noted that embryo cryo-preservation can reduce the occurrence of multiple pregnancies and preserve the fertility of cancer patients (2)(3)(4). However, little is known about its molecular impacts on embryos and the future newborne. Hence, considering molecular changes that may occur during and subsequent to embryo cryopreservation would provide a better picturefor decision making and managing probable undesirable Hsp72 Expression and Development of Vitrified 2-Cell Embryos outcomes. Evaluating the alterations of particular transcripts that may occur upon cryopreservation or global analysis of transcripts would be the first step towards answering some of the raised questions.
Cryopreservation of embryos usually can be performed either through slow freezing or vitrification. Commonly, a combination of high concentrations of cryoprotectants (typically dimethyl sulfoxide (DMSO) and ethylene glycol (EG) in addition to dehydrating agents such as sucrose or sorbitol have been used in vitrification. Extremely fast cooling of embryos via avoiding ice crystal formation allows vitrification to occur with minimum damage to the cells (4,5). However, use of high concentrations of cryoprotectants, which are often toxic to the cells may raise some questions regarding the safety issues of this technique (6).
Meanwhile, alterations of vitrification methodology can provide insights to reduce some of its drawbacks. These modifications can be performed either via increasing the cooling rate, a method known as ultra-rapid vitrification (7), or through reducing the vitrification solution volume. It has been suggested that even cells in pure water (without cryoprotectant) can be vitrified if the cooling rate is sufficiently high (8).
Expression of many genes including Heat shock protein (HSP) family, as its name indicates, is mainly affected in response to the changes in temperature (19). It was previously reported that the expression of Hsp72/Hsp73 is increased at the 2-cell stage (20). Accordingly, 2-cell mouse embryos were cryopreserved in the presence of two concentrations of cryoprotectants (30 and 15%) and subsequent changes of Hsp72 and Hprt1 (house-keeping gene) were analyzed upon thawing. Cryotop was the instrument of choice for vitrification. Vitrified and fresh 2-cell embryos were cultured to obtain cleavage and blastocyst formation rates. The aim of the study was to compare the effects of two different concentrations of cryoprotectants by cryotop vitrification on survival, developmental capacity and heat shock protein 72 (Hsp72) expression of two-cell mouse embryos.

Materials and Methods
This was an experimental study. This project was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences in 2009. All chemicals were purchased from Sigma Chemical (St Louis, MO, USA) unless it has been stated otherwise.
CD1 (ICR) female mice aged 8-10 weeks and male mice aged 10-12 weeks (Lisbon University, Portugal) were housed in polycarbonate cages (12 hours light/dark, 22 ± 2˚C), and were fed with standard food and fresh water. In all procedures, mice were handled according to the rules stipulated by the Animal Care in Portugal.

Study groups
The embryos were vitrified in two different concentrations of cryoprotectants by Cryotop and the changes of Hsp72 expression, survival, cleavage and blastocyst formation rates in vitrified and nonvitrified groups were assessed. The embryos from the mice sacrificed on each day were collected and then divided into two main groups, vitrified and control (non-vitrified) groups: the vitrified group was divided into two subgroups vit 1 (15% v/v: 7.5% DMSO+7.5% EG) and vit 2 (30% v/v: 15% DMSO+15% EG). Finally, 195 embryos of vitrified and control groups were evaluated for survival, cleavage and blastocyst rates. 200 embryos were assessed for expression of Hsp72 and Hprt1 as the reference gene (23,24).
For gene expression, each embryo pool containing 10 embryos was stored at -80˚C in a minimum volume (2 µl) of RNase free water (23). Experiments in each series were repeated at least three times.

Vitrification and thawing solutions
As the basal medium or washing solution (WS), modified Dulbecco's phosphate-buffered saline solution containing 10% (v/v) fetal bovine serum (GIBCO, CA, USA) was used. The equilibration solution contained 7.5% (v/v) EG and 7.5% (v/v) DMSO in basal medium.
There were two vitrification solutions (VS) for two vitrified groups, VS 1 :

Vitrification and thawing
Two concentrations of vitrification solutions were used to vitrify the mouse 2-cell embryos using Cryotop. Embryos of vit 1 and vit 2 groups were placed in three droplets of equilibration solution for 1 minute total for all of the drops at 25˚C. Subsequently, embryos were transferred into vitrification solution VS1 and VS2 respectively for less than 30 seconds. Embryos (6) were moved on the Cryotop (<1 μl vitrification solution) and the Cryotop was immediately submerged in filter-sterilized liquid nitrogen and kept for at least 7 days.
Samples were thawed by plunging the Cryotop into 1 ml of thawing solution at 37˚C for 1 minute. Rehydration and gradual removal of cryoprotectants were performed in D1, D2, D3, D4 and D5 for 3 minutes at every step. Thawed embryos were then washed three times in basal medium (Dul-becco's phosphate-buffered saline solution) for 5 minutes at 25˚C.

Definition of morphological surviv
Embryos were defined "morphologically survived", if the embryos possessed an intact zona pellucida, blastomeres and refractive cytoplasm (25,26). Following the thawing and cryoprotectant removal steps, embryos in 100 μl of sterilized KSOM+AA (Millipore, MA, USA) supplemented with 4 mg/ml BSA were incubated under mineral oil with the availability of 5% The validity of morphological classification was confirmed by vital staining with 0.4% sterilized trypan blue solution, a plasma membrane specific dye, in Hanks' balanced salt solution (HBSS) (27,28). The embryos were examined under an inverted micromanipulation microscope (Eppendorf, NY, USA). The dead cells were stained dark blue by trypan blue but viable cells were able to repel the dye and were not stained. They were counted and recorded as survival rates. Visually dead embryos were discarded, and the morphologically intact embryos were cultured and the gene expression pattern was analyzed.

Embryo culture
The survived embryos in control, vit 1 and vit 2 groups were cultured in 20 μl droplets of KSOM +AA supplemented with 4 mg/ml BSA under mineral oil at 37˚C in an atmosphere of 5% CO 2 , 5% O 2 and 90% N2 to develop into blastocysts. Embryos were assessed daily to record cleavage and blastocyst formation rates for 4 days.

Gene expression
The relative quantification of gene transcripts was carried out by real-time PCR. Super Script™ III Platinum ® Cells Direct Two-Step Quantitative reverse transcriptase PCR (qRT-PCR) Kit with SYBR ® Green (Invitrogen, CA, USA) was used to carry out cDNA synthesis and PCR.

Reverse transcription reaction
Embryos were lysed in 1 µl lysis enhancer and 10 µl resuspension buffer for every PCR tube, which were incubated at 75˚C for 10 minutes in a Thermal Cycler (Applied Biosystems 9700, CA, Hsp72 Expression and Development of Vitrified 2-Cell Embryos USA). To degrade any contaminating DNA, the cell lysates were treated with 5 µ1DNase I and 1.6 µl DNase I buffer (10×) at 25˚C for 5 minutes. The embryos were treated with 4 µl of 25-mM EDTA and incubated at 70˚C for 10 minutes. For first-Strand cDNA Synthesis, 20 µl 2× RT Reaction Mix and 2 µl RT Enzyme Mix were added to each tube which was then incubated at 25, 50 and 85˚C for 10, 20 and 5 minutes, respectively.

Nested quantitative polymerase chain reaction
Sometimes the expressions of some genes are very low, which makes the absolute quantification near to impossible. In such cases a prior polymerase chain reaction (PCR) amplification is required to boost the template level for the following quantification via Real-Time PCR, a technique called "nested quantitative PCR" or nqPCR for short (29,30). It is noteworthy to mention that the use of PCR amplicons instead of cDNA for the absolute quantification is not as accurate. However in places where the relative quantification serves the purpose, nqPCR provides enough accuracy. Additionally, considering the number of cells or the quantity of RNA that is used for cDNA synthesis, the expression level can be calculated.
The Primer pairs for each gene were designed, synthesized and validated by Molecular Diagnostic Companies (MDC, Burgess Hill, UK). The primer sequences, annealing temperatures and Gen Bank accession numbers are provided in table 1.

Amplicon size(bp)
Real-time PCR was conducted in a real-time cycler (Applied Biosystems 7500, CA, USA). To confirm the specificity and integrity of the PCR products, melting curve analyses were performed for all real-time PCR reactions. Standard curves were generated using serial dilutions of cDNAs. The cDNA of each sample was used as template for the preliminary PCR by Am-pliTaq Gold PCR Master Mix according to the manufacturer's instruction. Reactions were performed in a final volume of 50 μl. The firstround PCR mix contained 2 µl specific primer mix (300 nM), 25 µl master mix, 5 µl cDNA and 18 µl sterile water.
The first-round PCR was performed in a thermal cycler (Applied Biosystem 2720, California and USA), by incubation at 95˚C for 5 minutes, folalowed by 30 cycles of 95˚C for 15 seconds, specific Tm for every gene for 15 seconds (Table 1), and 72˚C for 60 seconds, and a final extension at 72˚C for 7 minutes. The PCR products were separated on 3% agarose gel (pure Nusieve GTC Agarose, Rockland, USA).
Cycling parameters were 50˚C for 2 minutes (UDG incubation), 95˚C for 2 minutes, followed by 50 cycles of 95˚C for 15 seconds and 60˚C for 30 seconds. The melting curve was analyzed at 95˚C for 15 sand temperature lowered to 60˚C for 15 seconds. Every experiment was repeated three times.
The data were analyzed with the integrated ABI 7500-V2.0.1 software (Applied Biosystem, California, USA) and were normalized with Hprt1 within the log linear phase of the amplification curve using the comparative Ct method (cycle threshold). The relative expression ratio (R) of Hsp72 was estimated based on a ΔCt formula (31)(32)(33). PCR efficiencies (32,33) of the genes ranged between 1.98-2.0. ΔCt was the difference between the Ct values of controls and samples.

Statistical analysis
One-way analysis of variance (ANOVA) was performed on the average percentages of survived, cleaved embryos, blastocyst formation and relative amount of Hsp72 mRNA in control, vit 1 and vit 2 groups. Following the analysis of variance, mean values were compared. The level of significance was set at less than 0.05 and least significant difference (LSD) test was used to compare treatments.

Developmental competence of 2-cell embryos following vitrification
In total, 195 in-vivo embryos at 2-cell stage were evaluated for survival, cleavage and blastocyst rates in control, vit 1 and vit 2 groups. The survival rates of vitrified and control groups are summarized in table 2, with no difference between vitrified groups and significantly lower than control (p< 0.05).

Control; Non-vitrified group, DMSO; Dimethyl sulfoxide and EG; Ethylene glycol, a and b indicate significant difference between control with vitrified groups (p<0.01). Every experiment was repeated three times.
The cleavage rates of embryos (2-cell to morula) in all groups are shown in figure 1. The cleavage rate in control (67.1% ± 1.6) was significantly higher than vit 1 (48.8% ± 0.9). Furthermore, the cleavage rate in vit 1 was significantly higher than vit 2 (36.8% ± 1.2) groups (p<0.05).  15% DMSO and 15% EG. a, b and c indicate the  significant differences among control, vit 1 and vit 2 (p<0.01).

Expression of Hsp72 mRNA
The effect of different concentrations of cryoprotectants on the expression of Hsp72 in 2-cell embryos was analyzed with nqPCR and the data were normalized against Hprt1. Hsp72 was significantly up-regulated, 12.9 fold in vit 1 and 32.4 fold in vit 2 , when compared to the control group (p<0.05, Fig 3). Moreover, the normalized relative expression ratio of Hsp72 in vit 2 was significantly higher than vit 1 (p<0.05).
Mean inverse Ct values of Hprt1 had no significant differences between vitrified and control groups (p>0.05, Fig 4).

Discussion
Mouse embryos can be cryopreserved efficiently at a wide range of developmental stages, 2-cell, 8-cell, or the morula stage (25,34). During the previous years, the success rates of vitrification have been improved by speeding up the cooling rates of cells via minimizing the sample size (vitrification solution and embryos). This increase effectively prohibits the ice crystal formation (1,25,35). Despite the fact that vitrification has proved to be useful in many aspects of cryobiology and fertility restoration, possible molecular consequences of vitrification are yet to be addressed properly. Initially, this can be ascertained through detailed molecular studies of genes that are directly involved in response to temperature change and stress response (36,37).
Association of Hsp70, Hsp27 and Hsp90 subfamilies have been demonstrated in the protection against apoptosis induced by a variety of stimuli such as heat shock, reactive oxygen species and cytoskeletal perturbation (38)(39)(40). Amongst the family of Hsp, Hsp72 is reported to be expressed at 2-cell embryos. For this reason, Hsp72 was considered as are presentative of the genes that maybe affected during vitrification with a variety of cryoprotectant concentrations. A concentration of a cryoprotectant is considered suitable when the expression pattern and morphological features of the fresh 2-cell embryos can be replicated as closeas possible. Indeed, this means that the cryoprotectant has had minimal effects on the cells.
Here, the previously proposed concentration of cryoprotectants (15% DMSO + 15% EG) was compared with the reduced concentration (7.5% DMSO +7.5% EG) in cryotop vitrification method. Ultimately, their effects on survival and developmental rates and on the expression of Hsp72 were compared with the control group (non-vitrification).
The results of the present study demonstrated that the survival rates were the same for both vitrification treatments, but the cleavage and blastocyst formation rates in vit 1 (our proposed concentration) were significantly higher than vit 2 for 2-cell mouse embryo. This may suggest reduced vitrification solution toxicity for vit 1 as opposed to vit 2 . Moreover, the survival and development rates of vitrified embryos were significantly lower than non-vitrified embryos. This might be due to the vitrification-thawing treatment of the embryos at an early stage of development and further be the result of poorly developed stress response mechanisms. In con-trast to our results, vitrification of human oocytes and embryos had no negative effect on survival and developmental rates (14,17,18). These dissimilar outcomes can be explained by the differences that are present between mice and human embryonic cells such as size and shape of the cells and membrane permeability (5).
Two other genes that were previously reported to be expressed in 2-cell embryos (41,42) were also considered for transcript analysis,Gja1 (Connexin 43), a gap junction gene (43)(44)(45), and Ped genes, a gene family regulating the rate of preimplantation embryonic development and subsequent embryo survival (46)(47)(48). However, our attempts to detect any expression of these genes at this stage failed (data not shown). Transcript analysis of Hsp72 showed an upregulation in vitrified groups when compared to the control group, similar to the previous results following other vitrification methods (25,49). Furthermore, the relative quantification of Hsp72 in vit 1 was significantly lower than vit 2 and closer to the fresh 2-cell embryos. The fact that Hsps play a protective role during imposed stresses to the cells, suppressing several forms of cell death, including apoptosis (50) may suggest that vit 1 treatment had a lesser impact on the overall well-being of the cell. In general, it can be said that 2-cell mouse embryos have experienced thermal stress during vitrification steps, but the concentrated cryoprotectants causes a pronounced stress to the embryos.

Conclusion
Our developmental data show that cryotopvitrification with 7.5% EG and 7.5% DMSO was more efficient than that with 15% EG and 15% DMSO. Although vit 1 treatment had lower survival and developmental rates compared to the control group, it demonstrated better stability compared with vit 2 based on the Hsp72 transcript analysis, supporting developmental data.